Human male reproductive development is a highly organized and regulated process that is susceptible to environmental toxicants such as endocrine-disrupting chemicals and medical treatments. Environmental influences and insults by reproductive toxicant exposure during early development or post-puberty can lead to impaired spermatogenesis or, at worst, infertility. Understanding how certain chemicals disrupt spermatogenesis during various Windows of Susceptibility (WOS) is critical for determining how environmental and industrial toxicants contribute to impaired fertility. Furthermore, these toxicants can potentially induce epigenetic alterations that contribute to disease phenotypes in offspring and thus have transgenerational consequences such as increased risk of producing offspring with neurological disorders, cardiac malformations, limb defects, and other developmental deficiencies that lead to debilitating disorders. To date, the most widely used models for exploring the effects of environmental factors on reproductive toxicity and multigenerational defects are rodent models. While informative, these models poorly recapitulate human spermatogenesis as rodent spermatogenesis proceeds in a distinctly different fashion compared to human spermatogenesis. Thus, developing human in vitro models for reproductive toxicity testing is paramount for advancing the field and for understanding WOS as well as the cellular and molecular mechanisms that may underlie these disruptions in human spermatogenesis. This proposal seeks to utilize my recently published, novel in vitro model of human spermatogenesis to examine the effects and identify sub-cellular mechanisms of known and unknown/predicted reproductive toxicants on various windows of susceptibility during spermatogenesis.